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1.
Mol Cell ; 65(2): 361-370, 2017 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-28065596

RESUMO

Targeted mass spectrometry assays for protein quantitation monitor peptide surrogates, which are easily multiplexed to target many peptides in a single assay. However, these assays have generally not taken advantage of sample multiplexing, which allows up to ten analyses to occur in parallel. We present a two-dimensional multiplexing workflow that utilizes synthetic peptides for each protein to prompt the simultaneous quantification of >100 peptides from up to ten mixed sample conditions. We demonstrate that targeted analysis of unfractionated lysates (2 hr) accurately reproduces the quantification of fractionated lysates (72 hr analysis) while obviating the need for peptide detection prior to quantification. We targeted 131 peptides corresponding to 69 proteins across all 60 National Cancer Institute cell lines in biological triplicate, analyzing 180 samples in only 48 hr (the equivalent of 16 min/sample). These data further elucidated a correlation between the expression of key proteins and their cellular response to drug treatment.


Assuntos
Ensaios de Triagem em Larga Escala , Espectrometria de Massas , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Proteoma , Proteômica/métodos , Antibióticos Antineoplásicos/farmacologia , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Fatores de Tempo , Fatores de Transcrição/metabolismo , Fluxo de Trabalho
2.
Nat Methods ; 15(9): 732-740, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30127506

RESUMO

Human embryonic stem cells (hESCs) can be captured in a primed state in which they resemble the postimplantation epiblast, or in a naive state where they resemble the preimplantation epiblast. Naive-cell-specific culture conditions allow the study of preimplantation development ex vivo but reportedly lead to chromosomal abnormalities, which compromises their utility in research and potential therapeutic applications. Although MEK inhibition is essential for the naive state, here we show that reduced MEK inhibition facilitated the establishment and maintenance of naive hESCs that retained naive-cell-specific features, including global DNA hypomethylation, HERVK expression, and two active X chromosomes. We further show that hESCs cultured under these modified conditions proliferated more rapidly; accrued fewer chromosomal abnormalities; and displayed changes in the phosphorylation levels of MAPK components, regulators of DNA damage/repair, and cell cycle. We thus provide a simple modification to current methods that can enable robust growth and reduced genomic instability in naive hESCs.


Assuntos
Células-Tronco Embrionárias/metabolismo , Instabilidade Genômica , MAP Quinase Quinase Quinases/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Metilação de DNA , Células-Tronco Embrionárias/enzimologia , Humanos , Proteoma , Transcriptoma
3.
J Proteome Res ; 18(3): 1299-1306, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30658528

RESUMO

Quantitative proteomics employing isobaric reagents has been established as a powerful tool for biological discovery. Current workflows often utilize a dedicated quantitative spectrum to improve quantitative accuracy and precision. A consequence of this approach is a dramatic reduction in the spectral acquisition rate, which necessitates the use of additional instrument time to achieve comprehensive proteomic depth. This work assesses the performance and benefits of online and real-time spectral identification in quantitative multiplexed workflows. A Real-Time Search (RTS) algorithm was implemented to identify fragment spectra within milliseconds as they are acquired using a probabilistic score and to trigger quantitative spectra only upon confident peptide identification. The RTS-MS3 was benchmarked against standard workflows using a complex two-proteome model of interference and a targeted 10-plex comparison of kinase abundance profiles. Applying the RTS-MS3 method provided the comprehensive characterization of a 10-plex proteome in 50% less acquisition time. These data indicate that the RTS-MS3 approach provides dramatic performance improvements for quantitative multiplexed experiments.


Assuntos
Peptídeos/isolamento & purificação , Proteoma/isolamento & purificação , Proteômica/métodos , Algoritmos , Bases de Dados Factuais , Humanos , Peptídeos/química , Proteoma/química , Espectrometria de Massas em Tandem , Fluxo de Trabalho
4.
J Proteome Res ; 16(2): 1069-1076, 2017 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-27978624

RESUMO

While developing a multiplexed phosphotyrosine peptide quantification assay, an unexpected observation was made: significant neutral loss from phosphotyrosine (pY) containing peptides. Using a 2000-member peptide library, we sought to systematically investigate this observation by comparing unlabeled peptides with the two highest-plex isobaric tags (iTRAQ8 and TMT10) across CID, HCD, and ETD fragmentation using high resolution high mass accuracy Orbitrap instrumentation. We found pY peptide neutral loss behavior was consistent with reduced proton mobility, and does not occur during ETD. The site of protonation at the peptide N-terminus changes from a primary to a tertiary amine as a result of TMT labeling which would increase the gas phase basicity and reduce proton mobility at this site. This change in fragmentation behavior has implications during instrument method development and interpretation of MS/MS spectra, and therefore ensuing follow-up studies. We show how sites not localized to tyrosine by search and site localization algorithms can be confidently reassigned to tyrosine using neutral loss and phosphotyrosine immonium ions. We believe these findings will be of general interest to those studying pY signal transduction using isobaric tags.


Assuntos
Peptídeos/química , Fosfotirosina/química , Espectrometria de Massas por Ionização por Electrospray/normas , Peptídeos/análise , Fosfotirosina/análise , Kit de Reagentes para Diagnóstico , Coloração e Rotulagem/métodos , Tirosina/química
5.
PLoS Biol ; 11(8): e1001637, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23976882

RESUMO

The human gut microbiota is an important metabolic organ, yet little is known about how its individual species interact, establish dominant positions, and respond to changes in environmental factors such as diet. In this study, gnotobiotic mice were colonized with an artificial microbiota comprising 12 sequenced human gut bacterial species and fed oscillating diets of disparate composition. Rapid, reproducible, and reversible changes in the structure of this assemblage were observed. Time-series microbial RNA-Seq analyses revealed staggered functional responses to diet shifts throughout the assemblage that were heavily focused on carbohydrate and amino acid metabolism. High-resolution shotgun metaproteomics confirmed many of these responses at a protein level. One member, Bacteroides cellulosilyticus WH2, proved exceptionally fit regardless of diet. Its genome encoded more carbohydrate active enzymes than any previously sequenced member of the Bacteroidetes. Transcriptional profiling indicated that B. cellulosilyticus WH2 is an adaptive forager that tailors its versatile carbohydrate utilization strategy to available dietary polysaccharides, with a strong emphasis on plant-derived xylans abundant in dietary staples like cereal grains. Two highly expressed, diet-specific polysaccharide utilization loci (PULs) in B. cellulosilyticus WH2 were identified, one with characteristics of xylan utilization systems. Introduction of a B. cellulosilyticus WH2 library comprising >90,000 isogenic transposon mutants into gnotobiotic mice, along with the other artificial community members, confirmed that these loci represent critical diet-specific fitness determinants. Carbohydrates that trigger dramatic increases in expression of these two loci and many of the organism's 111 other predicted PULs were identified by RNA-Seq during in vitro growth on 31 distinct carbohydrate substrates, allowing us to better interpret in vivo RNA-Seq and proteomics data. These results offer insight into how gut microbes adapt to dietary perturbations at both a community level and from the perspective of a well-adapted symbiont with exceptional saccharolytic capabilities, and illustrate the value of artificial communities.


Assuntos
Bacteroides/genética , Bacteroides/metabolismo , Trato Gastrointestinal/microbiologia , Microbiota/fisiologia , Animais , Genoma Bacteriano/genética , Humanos , Masculino , Camundongos , Microbiota/genética , Simbiose
6.
Microbiome ; 7(1): 18, 2019 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-30744677

RESUMO

BACKGROUND: The gut microbiome plays a fundamental role in the human host's overall health by contributing key biological functions such as expanded metabolism and pathogen defense/immune control. In a healthy individual, the gut microbiome co-exists within the human host in a symbiotic, non-inflammatory relationship that enables mutual benefits, such as microbial degradation of indigestible food products into small molecules that the host can utilize, and enhanced pathogen defense. In abnormal conditions, such as Crohn's disease, this favorable metabolic relationship breaks down and a variety of undesirable activities result, including chronic inflammation and other health-related issues. It has been difficult, however, to elucidate the overall functional characteristics of this relationship because the microbiota can vary substantially in composition for healthy humans and possibly even more in individuals with gut disease conditions such as Crohn's disease. Overall, this suggests that microbial membership composition may not be the best way to characterize a phenotype. Alternatively, it seems to be more informative to examine and characterize the functional composition of a gut microbiome. Towards that end, this study examines 25 metaproteomes measured in several Crohn's disease patients' post-resection surgery across the course of 1 year, in order to examine persistence of microbial taxa, genes, proteins, and metabolic functional distributions across time in individuals whose microbiome might be more variable due to the gut disease condition. RESULTS: The measured metaproteomes were highly personalized, with all the temporally-related metaproteomes clustering most closely by individual. In general, the metaproteomes were remarkably distinct between individuals and to a lesser extent within individuals. This prompted a need to characterize the metaproteome at a higher functional level, which was achieved by annotating identified protein groups with KEGG orthologous groups to infer metabolic modules. At this level, similar and redundant metabolic functions across multiple phyla were observed across time and between individuals. Tracking through these various metabolic modules revealed a clear path from carbohydrate, lipid, and amino acid degradation to central metabolism and finally the production of fermentation products. CONCLUSIONS: The human gut metaproteome can vary quite substantially across time and individuals. However, despite substantial intra-individual variation in the metaproteomes, there is a clear persistence of conserved metabolic functions across time and individuals. Additionally, the persistence of these core functions is redundant across multiple phyla but is not always observable in the same sample. Finally, the gut microbiome's metabolism is not driven by a set of discrete linear pathways but a web of interconnected reactions facilitated by a network of enzymes that connect multiple molecules across multiple pathways.


Assuntos
Bactérias/metabolismo , Doença de Crohn/microbiologia , Microbioma Gastrointestinal/fisiologia , Proteoma/metabolismo , Acetilglucosamina/análise , Adulto , Bactérias/genética , Doença de Crohn/cirurgia , Ácido N-Acetilneuramínico do Monofosfato de Citidina/análise , Ácidos Graxos Voláteis/análise , Fezes/microbiologia , Feminino , Microbioma Gastrointestinal/genética , Humanos , Masculino , Pessoa de Meia-Idade , Proteômica , RNA Ribossômico 16S/genética
7.
Cell Syst ; 8(5): 412-426.e7, 2019 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-31078528

RESUMO

Tyrosine kinase inhibitors (TKIs) are widely used to treat solid tumors but can be cardiotoxic. The molecular basis for this toxicity and its relationship to therapeutic mechanisms remain unclear; we therefore undertook a systems-level analysis of human cardiomyocytes (CMs) exposed to four TKIs. CMs differentiated from human induced pluripotent stem cells (hiPSCs) were exposed to sunitinib, sorafenib, lapatinib, or erlotinib, and responses were assessed by functional assays, microscopy, RNA sequencing, and mass spectrometry (GEO: GSE114686; PRIDE: PXD012043). TKIs have diverse effects on hiPSC-CMs distinct from inhibition of tyrosine-kinase-mediated signal transduction; cardiac metabolism is particularly sensitive. Following sorafenib treatment, oxidative phosphorylation is downregulated, resulting in a profound defect in mitochondrial energetics. Cells adapt by upregulating aerobic glycolysis. Adaptation makes cells less acutely sensitive to sorafenib but may have long-term negative consequences. Thus, CMs exhibit adaptive responses to anti-cancer drugs conceptually similar to those previously shown in tumors to mediate drug resistance.


Assuntos
Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Aclimatação , Antineoplásicos/farmacologia , Cardiotoxicidade/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Cloridrato de Erlotinib/farmacologia , Perfilação da Expressão Gênica/métodos , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Lapatinib/farmacologia , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sorafenibe/farmacologia , Sunitinibe/farmacologia
8.
J Am Soc Mass Spectrom ; 29(7): 1505-1511, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29671274

RESUMO

Phosphorylation-mediated signaling pathways have major implications in cellular regulation and disease. However, proteins with roles in these pathways are frequently less abundant and phosphorylation is often sub-stoichiometric. As such, the efficient enrichment, and subsequent recovery of phosphorylated peptides, is vital. Mass spectrometry-based proteomics is a well-established approach for quantifying thousands of phosphorylation events in a single experiment. We designed a peptide internal standard-based assay directed toward sample preparation strategies for mass spectrometry analysis to understand better phosphopeptide recovery from enrichment strategies. We coupled mass-differential tandem mass tag (mTMT) reagents (specifically, TMTzero and TMTsuper-heavy), nine mass spectrometry-amenable phosphopeptides (phos9), and peak area measurements from extracted ion chromatograms to determine phosphopeptide recovery. We showcase this mTMT/phos9 recovery assay by evaluating three phosphopeptide enrichment workflows. Our assay provides data on the recovery of phosphopeptides, which complement other metrics, namely the number of identified phosphopeptides and enrichment specificity. Our mTMT/phos9 assay is applicable to any enrichment protocol in a typical experimental workflow irrespective of sample origin or labeling strategy. Graphical Abstract ᅟ.


Assuntos
Metais/química , Óxidos/química , Fosfopeptídeos/análise , Fosfopeptídeos/isolamento & purificação , Espectrometria de Massas em Tandem/métodos
9.
PLoS One ; 7(11): e49138, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23209564

RESUMO

Crohn's disease (CD) is an inflammatory bowel disease of complex etiology, although dysbiosis of the gut microbiota has been implicated in chronic immune-mediated inflammation associated with CD. Here we combined shotgun metagenomic and metaproteomic approaches to identify potential functional signatures of CD in stool samples from six twin pairs that were either healthy, or that had CD in the ileum (ICD) or colon (CCD). Integration of these omics approaches revealed several genes, proteins, and pathways that primarily differentiated ICD from healthy subjects, including depletion of many proteins in ICD. In addition, the ICD phenotype was associated with alterations in bacterial carbohydrate metabolism, bacterial-host interactions, as well as human host-secreted enzymes. This eco-systems biology approach underscores the link between the gut microbiota and functional alterations in the pathophysiology of Crohn's disease and aids in identification of novel diagnostic targets and disease specific biomarkers.


Assuntos
Doença de Crohn/metabolismo , Doença de Crohn/microbiologia , Metagenoma , Metagenômica , Proteômica , Bactérias/genética , Bactérias/metabolismo , Análise por Conglomerados , Feminino , Trato Gastrointestinal/metabolismo , Trato Gastrointestinal/microbiologia , Humanos , Íleo/metabolismo , Íleo/microbiologia , Íleo/patologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Masculino , Redes e Vias Metabólicas , Proteoma , Gêmeos Monozigóticos
10.
PLoS One ; 6(11): e27173, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22132090

RESUMO

Accurate protein identification in large-scale proteomics experiments relies upon a detailed, accurate protein catalogue, which is derived from predictions of open reading frames based on genome sequence data. Integration of mass spectrometry-based proteomics data with computational proteome predictions from environmental metagenomic sequences has been challenging because of the variable overlap between proteomic datasets and corresponding short-read nucleotide sequence data. In this study, we have benchmarked several strategies for increasing microbial peptide spectral matching in metaproteomic datasets using protein predictions generated from matched metagenomic sequences from the same human fecal samples. Additionally, we investigated the impact of mass spectrometry-based filters (high mass accuracy, delta correlation), and de novo peptide sequencing on the number and robustness of peptide-spectrum assignments in these complex datasets. In summary, we find that high mass accuracy peptide measurements searched against non-assembled reads from DNA sequencing of the same samples significantly increased identifiable proteins without sacrificing accuracy.


Assuntos
Bactérias/metabolismo , Ecossistema , Metagenômica/métodos , Proteômica/métodos , Características de Residência , Sequência de Aminoácidos , Bases de Dados de Proteínas , Feminino , Humanos , Peptídeos/metabolismo , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos
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